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Topic: Background Reduction (Read 8264 times)

Hi guys, I'm a new poster, I have a question about background autoflourecence in regards to negative controls.

I am doing a project staining bone marrow sections with Alexaflour 488 as secondary. My analysis segment is done using a manual count based on visual differentiation between 'light' and 'dark' cell areas. Under FITC light I've noticed that my negative controls, both flourescence secondary only and mouse isotype antibody are definitely darker in general but also show some signs of marker flourescence though not as bright as my experimental group images.

I have two questions:

1) What is causing the flourescent patchy cell areas in the negatives?2) How can I better differentiate between auto and intended flourescence?

If someone could show me how i could post some example images on the site to better illustrate my issue.

Are you dealing with normal or diseased tissues? For one of my colleagues, when he was doing antibody optimisation he experienced terrible terrible background using normal mouse spleen and lymph nodes. He was advised to use 0.1 M HCl to block the endogenous alkaline phosphatase and it got rid of the background completely! However, it destroyed almost all of the antigen epitodes for some of the cell markers that he was staining for (e.g. CD4 and foxp3). Since then I've removed that step. I used normal mouse colon and there was absolutely no background! I think it could have to do with inadequate washing. Also if yours tissues get dried up during incubation times it tends to result in staining artefacts, which I've also experienced! Nevertheless, when I apply the exact protocol to colitis tissues, I had really bad background. My team said it might to due to storage issues - repeated freezing and thawing the slides (freezing artefact). For your second question, you should browse through the literature and look for papers that more or less doing the same thing as you.

Definitely post up some pictures, it will make it mush easier for us to give suggestions. Hope this helps!